For over thirty years, isoelectric focusing (IEF) has served as a primary tool for analyzing proteins present in complex admixture, such as proteins present in biological samples.
In isoelectric focusing, proteins are driven by an applied electric field through a pH gradient typically established in a support matrix, such as a gel. Proteins migrate until the isoelectric point (pI) of the protein coincides with the local pH; at that point, the protein no longer bears net charge and ceases to migrate, becoming focused at a point that is characteristic of the protein.
As originally described, the pH gradient for IEF was established and sustained in the gel matrix by mobile carrier ampholytes (CA). Gels typically would be polymerized in the presence of a population of CA having a range of charge characteristics; upon application of a voltage gradient, the various species of CA would align themselves in the matrix to establish a pH gradient across the gel.
Although IEF with CA has proven tremendously useful, it was soon discovered that pH gradients created by CA were susceptible to titration by atmospheric carbon dioxide, leading to the migration of CA towards the cathode and destruction of the pH gradient over time, a phenomenon termed cathodic drift.
Cathodic drift can be reduced by casting IEF gels in enclosed tubes, thus limiting exposure to atmospheric CO2. However, the tube traps prepolymer component impurities in the matrix during polymerization, interfering with separation. Furthermore, the tube format presents difficulties when a second dimension of separation, such as fractionation by size, is desired.
In a different approach to the problem of cathodic drift, Bjellqvist and colleagues immobilized the pH gradient in the support matrix, an approach now termed immobilized pH gradient (IPG) isoelectric focusing. See Bjellqvist et al., J. Biochem. Biophys. Methods 6(4):317-39 (1982); Righetti et al., Trends Biochem. Sci. 13(9):335-8 (1988); Righetti et al., Methods Enzymol. 270:235-55 (1996); U.S. Pat. No. 4,130,470; and Righetti, Immobilized pH Gradient: Theory and Methodology, (Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 20), Elsevier Biomedical Press, LTD, Netherlands (ASIN: 0444813012). Two-dimensional electrophoresis, with IPG IEF followed by size fractionation, soon followed. Gorg et al., Electrophoresis 9(9):531-46 (1988).
IPG not only reduced the problem of cathodic drift, but also proved useful in reducing interference from prepolymer component impurities, since the IPG strip's plastic backing imparts sufficient structural resilience to the gel as to permit the gel to be washed before use. The increased resilience also permits the gels to be stored in dehydrated form before use. Dehydrated IPG strips are today sold in a variety of pH ranges and a variety of separation lengths by a number of vendors (e.g., Immobiline DryStrip Gels, Amersham Biosciences, Piscataway, N.J., USA; ReadyStrip IPG, Bio-Rad Laboratories, Hercules, Calif., USA).
Problems Remain, However.
Although immobilization of the gradient-forming ampholytes prevents cathodic drift, the charge-bearing immobilized moieties (immobilines) remain susceptible to titration by atmospheric CO2. CO2 titration is exacerbated by the fact that the separation medium of IPG strips is directly exposed to air on at least one side. Direct exposure to air also leads to possible dehydration of the matrix, with possible salt crystallization, during electrophoresis.
These problems have been addressed in part by a methodological, rather than structural, solution: plastic-backed IPG strips are typically electrophoresed under an occlusive oil layer, which both excludes air and retards evaporation.
Use of an occlusive liquid oil layer presents its own difficulties, however. Principal among these is the requirement that electrophoresis be performed with the IPG strip maintained in a horizontal orientation. The obligate horizontal orientation precludes use of the smaller-footprint, vertical electrophoresis devices typically used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), such as those described in Tippins et al., U.S. Pat. No. 5,888,369. In addition, the use of oil requires deft manual technique and proves time-intensive.
Wiktorowicz et al., U.S. Pat. No. 6,013,165, describe an apparatus in which immobilized pH gradient isoelectric focusing can be performed without use of a liquid oil layer. A continuous pKa gradient is immobilized on at least one of the major opposing surfaces of a cavity formed between two plates. The cavity, which can be further segmented into parallel channels, is then filled with a flowable separation medium. Electrophoresis is preferably conducted with the assembly oriented horizontally to minimize convection currents in the flowable separation medium. The apparatus does not readily permit insertion of prior-cast hydratable separation media, such as commercial IPG strips, nor does it readily permit electrophoresis in the vertical dimension.
There thus exists a need in the art for methods and apparatus that allow IPG strips, and other prior-cast hydratable separation media, to be electrophoresed without requiring contact with an occlusive fluid oil layer. There further exists a need in the art for methods and apparatus that allow IPG strips, and other prior-cast hydratable separation media, to be electrophoresed in a vertical orientation.